This invention relates to novel man-made bacterial strains which produce chitinase, an enzyme which degrades chitin. This invention further relates to the use of such strains as a means to inhibit soil fungi and nematodes and to enhance plant growth by biological control of plant pathogens. This invention additionally relates to the introduction of chitinase activity into plants and to plants which have been rendered resistant to plant pathogens as a result of such introduction, and to plants which have been rendered resistant to chilling or frost (freezing) damage as a result of such introduction. Finally, this invention relates to the introduction into plant cells of a sequence from the chitinase gene that directs the secretion of polypeptides encoded by foreign DNA.
The soil contains a wide variety of life forms which can interact with plants, including bacteria, fungi and nematodes. These life forms are especially abundant in the rhizosphere, the area of the soil that surrounds and is influenced by the plant roots. As used herein the term rhizosphere embraces the rhizoplane, the root-soil interface including the surface of the root. The term rhizobacteria, as used herein, refers to bacteria adapted to the rhizosphere. The interactions between these soil inhabiting life forms are complex, some being antagonistic and others being mutually beneficial.
The interactions between plants and the various soil life forms are similarly complex, in some instances helpful to the plant and in other instances deleterious to the plant. Fungi harmful to plants (fungal pathogens) include fungal species from a wide variety of genera, including Fusarium, Pythium, Phytophthora, Verticillium, Rhizoctonia, Macrophomina, Thielaviopsis, Sclerotinia and numerous others. Plant diseases caused by fungi include pre and post-emergence seedling damping-off, hypocotyl rots, root rots, crown rots, vascular wilts and a variety of other forms of symptom development. Nematodes harmful to plants (nematode pathogens) include nematode species from the genera Meloidogyne, Heterodera, Ditylenchus, Pratylenchus. Plant diseases caused by nematodes include root galls, root rot, lesions, "stubby" root, stunting, and various other rots and wilts associated with increased infection by pathogenic fungi. Some nematodes (e.g., Trichodorus, Lonaidorus, Xiphenema) can serve as vectors for virus diseases in a number of plants including Prunus, grape, tobacco and tomato.
Various approaches are available for attempting to control deleterious fungi and nematodes. One method, long known in the art, is chemical treatment of soil or plants with fungicides or nematicides. Another method is application of certain naturally occurring bacteria which inhibit or interfere with fungi or nematodes. See, in general, K. F. Baker and R. J. Cook, Biological Control of Plant Pathogens, Freeman and Co. (1974) for a description of fungi and nematodes and their interaction with plants, as well as a description of means for biological control of fungal and nematode pathogens.
One approach to biocontrol of fungal and nematode pathogens is based on the widespread presence of chitin as an integral part of the cell walls of fungi and the outer covering of nematodes or nematode eggs or nematode cysts. Chitin is an unbranched polysaccharide polymer consisting of N-acetyl-D-glucosamine units ("GluNAc") joined by beta-1,4 glycosidic linkages. Chitin is insoluble in water, dilute mineral acids and bases but can be broken down enzymatically by chitinase, the degradation products being soluble monomers or multimers of GluNAc. Chitinases are a class of hydrolytic enzymes which degrade chitin by endolytic or exolytic mechanisms. The endochitinases, e.g., many of the chitinases found in plants, cleave the internal beta-1,4 glycosidic linkages in the chitin molecules to liberate oligomers of at least 3 GluNAc units. The exochitinases, e.g., bacterial chitinases such as present in Serratia marcescens hydrolyze beta-1,4 glycosidic bonds and release GluNAc; see Roberts et al., "Plant and Bacterial Chitinases Differ in Antifungal Activity", (1988) J. Gen. Microbiol. 134, 169-176, and references cited therein. Chitinase is produced by certain naturally occurring bacteria, fungi, nematodes, insects, crustaceans, plants and some vertebrates and there have been reports of the role of chitinase in the suppression of pathogens.
R. Mitchell and M. Alexander, "The Mucolytic Phenomenon and Biological Control of Fusarium in Soil", Nature, 190, 109-110 (1961) describes naturally occurring mucolytic, or fungi-lysing, soil bacteria (genera Bacillus and Pseudomonas) which suppress soil Fusarium by means of chitinase activity. B. Sneh, "Use of Rhizosphere Chitinolytic Bacteria for Biological Control", Phytopath. Z., 100, 251-56 (1981) discloses naturally occurring chitinolytic isolates identified as Arthrobacter sp. and Serratia liquifaciens. Sneh also discloses introduction of a chitinolytic bacterial strain from the genus Arthrobacter into the rhizosphere to protect carnation seedlings from Fusarium wilt.
A. H. Michael and P. E. Nelson, "Antagonistic effect of soil bacteria on Fusarium roseum culmorum", Phytopathology, 62, 1052-1056 (1972) discloses similar control with a naturally occurring Pseudomonas species.
J. Monreal and E. T. Reese, "The Chitinase of Serratia marcescens", Canadian Journal of Microbiology, 15, 689-696 (1969) describes a Serratia marcescens bacterial strain (QMBl466) selected as the most active chitinase producer out of a number of naturally occurring bacterial and fungal strains tested. Other strains tested which displayed some chitinase activity included bacterial strains from the genera Enterobacter and Streptomyces, and fungal strains from the genera Aspergillus, Penicillium and Trichoderma. Chitinase is characterized as an induced enzyme system in strain QMBl466, i.e., the yields of chitinase produced by the strain were higher when chitin was present. Monreal et al. reports at p. 692 that chitinase production on a chitin medium is repressed by the addition of other carbon-containing metabolites, e.g., sugars, to the medium. The Serratia marcescens enzyme system is described as extracellular and including endochitinase, a chitobiase and a "factor" for hydrolysis of "crystalline" chitin.
The naturally occurring Serratia marcescens chitinase system is further described in R. L. Roberts and E. Cabib, "Serratia Marcescens Chitinase: One-Step Purification and Use for the Determination of Chitin", Analytical Biochemistry, 127, 402-412 (1982).
J. D. Reid and D. M. Ogrydziak, "Chitinase-Overproducing Mutant of Serratia marcescens", Applied and Environmental Microbiology, 41, 664-669 (1981) describes work with a mutant of Serratia marcescens, strain IMR-lEl, obtained by mutation of strain QMBl466. The mutant had increased chitinase activity compared to strain QMBl466, as measured by zones of clearing on chitin-agar plates. On page 664 Reid et al. refers to the "high rate of reversion of IMR-1E1 to decreased levels of chitinase production."
C. I. Kado and P. F. Lurguin, "Prospectus for Genetic Engineering in Agriculture", Phytopathogenic Prokaryotes, Vol. 2, M. S. Mount and G. H. Lacy eds., 309 (1982), while not discussing the role of chitinase in controlling chitin-containing pathogens, notes the possibility of a different approach to controlling fungi, namely, inserting into bacteria genes coding for compounds which inhibit chitin synthase in fungi. That is, the compound chitin synthase, necessary for production of chitin in fungi, would be inhibited by the bacterial compounds.
P. M. Miller and D. C. Sands, "Effects of Hydrolytic Enzymes on Plant-parasitic Nematodes", Journal of Nematology, 9, 192-197 (1977) describes the effect of chitinase, obtained from a commercial supplier, on certain nematodes. Miller et al. discloses that chitinase hydrolytic enzymes are toxic to certain nematodes, in particular Tylenchorhynchus dubius, the toxicity being greater in aqueous solution than in soil.
There are a number of limiting factors and disadvantages with respect to work to date on biological control of plant pathogens using chitinase-producing bacteria introduced into the soil. First is the inability to regulate the production of chitinase in the introduced bacteria in such a way that proper amounts of chitinase are produced. Second is the limited ability of many of such bacteria to colonize and persist in the rhizosphere of host plants, a key consideration for effective biocontrol. Particularly important in this respect is the ability of biocontrol bacteria to colonize the roots of host plants effectively, the roots being the site of much plant-pathogen interaction. Third is that chitinase production is repressed in the presence of other carbon sources, e.g., metabolites released by the root. Another problem, at least as to mutants, is reversion to forms exhibiting decreased levels of chitinase production. There have been a number of reports of methods for introducing foreign DNA into plants. One approach is introduction by transformation using Agrobacterium, in particular Agrobacterium tumefaciens; M. Bevan et al., Ann. Rev. Genet., 16, 357-384 (1982); L. Ream et al., Science, 218, 854-859 (1982). This introduction may be carried out by cocultivation of plant protoplasts with Agrobacterium, followed by plant regeneration; Marton et al., Nature, 277, 129-131 (1979); R. B. Horsch et al., Science, 223, 496-498 (1984). The introduction may also be carried out using binary vectors; A. Hoekema et al., Nature, 303, 179-180 (1983); P. van den Elzen et al., Plant Mol. Biol., 5, 149-154 (1985); M. Bevan, Nucl. Acids Res., 12, 8711-8721 (1984). Where the foreign DNA is from a non-plant source, the foreign DNA (structural gene, i.e., encoding sequence) may be fused to a plant promoter; L. Herrera-Estrella et al. Nature, 303, 209-213 (1983); R. T. Fraley et al., Proc. Natl. Acad. Sci., 80, 4803-4807 (1983); J. T. Odell et al., Nature, 313, 810-812 (1985); J. Jones et al., EMBO J., 4, 2411-2418 (1985).